The design, synthesis and application of a new type of fluorescent biological probe is proposed. Photoactivable fluorophores (PAFs) will possess latent fluorescent properties that can be revealed by photochemical activation. Activation of PAFs attached to cellular components or antibody complexes will selectively switch """"""""on"""""""" fluorescence at any arbitrarily designated time and in a spatially well-defined cellular compartment or location. PAF's will enable studies of cellular dynamics, motility and translocation phenomena, now difficult or impossible using conventional fluorescence techniques or other tracer methodology. Several fundamentally different types of PAFs will be prepared, each based on a different photoinitiated chemical sequence to convert the non-fluorescent PAFs to ultimate fluorophores. Deactivating or quenching protecting groups, removable by photochemical reaction, will be integral components of one type of PAF. Photostimulated nucleophilic aromatic substitution, photoinduced stilbene type cyclizations, and photo chemically generated nitrenes and carbenes, which can readily cyclize by intramolecular C-H bond insertions, will provide the mechanistic bases for other types of PAFs. Studies will be conducted to determine kinetics, efficiency and wavelength dependence of the photoactivation process and to evaluate fluorescence characteristics of the PAFs and photogenerated fluorescent molecules. Purified actin will be labeled with a suitably functionalized PAF, and employed in the development of new fluorescence technique. The new """"""""fluorescence dissipation"""""""" technique (to be developed in collaboration with Professor Ben Ware) will be a highly sensitive replacement for fluorescence photobleaching recovery (FPR) methods in studies of actin assembly. This new technique will be a generally applicable replacement for FPR methods in many other types of investigations. Photoactivable fluorophores will represent an innovative technology, capable of revolutionizing existing fluorescence-based studies and breaking new ground in emerging biophysical and biochemical investigations of significant relevance to fundamental biomedical research.
